1.Field of the Invention
This invention relates generally to gene expression systems comprising an expression vector and a "trans-acting DNA segment" where the expression vector comprises the gene or genes to be expressed and one or more cis-acting regulatory elements which are responsive to a trans-acting factor produced by said "trans-acting DNA segment". More specifically the invention relates to such gene expression systems where said "trans-acting DNA segment" and said cis-acting regulatory elements comprise one or more segments of the genome from a Bacillus species.
2. Description of the Background Art
Gene expression systems, where the expression of a specific gene is regulated in the transcriptional phase by the binding of a repressor molecule to the DNA sequence comprising the operator for the gene concerned, thus preventing RNA polymerase from binding to the promotor, and in which induction occurs through the binding of an inducer to the repressor molecule, which causes a conformational shift in the repressor, decreasing the affinity of the repressor for the operator, are well known in Eschericia coli.
Such operator-repressor control systems as for example the lac operon therefore have for a number of years been used for the expression of foreign genes in E. coli.
Recently an interest has been raised for the cloning and expression of foreign genes in bacilli, especially Bacillus subtilis, for a number of reasons. In contrast to E coli, B. subtilis is not a pathogen, and it does not produce endotoxins. Also it possesses a secretory system that releases proteins into the culture medium in large quantities. Its genetic map is well characterized, and a great deal of information on large-scale cultivation of B. subtilis is available since the organism is widely used in industry. One disadvantage of B. subtilis strains, the formation of spores, has been circumvented by the development of asporogenic B. subtilis strains.
The gene expression systems hitherto known in bacilli that work on the transcriptional level are, however, not regulated by induction, but use a different mechanism. Gene regulation in these organisms is controlled by .sigma.-factors, which are proteins that bind to the RNA polymerase and determine the recognition site for RNA initiation. Such systems cannot easily be used for the controlled expression of a foreign gene.
The attempts that have been made in expressing foreign genes in B. subtilis have until now concentrated on using .alpha.-amylase derived secretion vectors (Palva et al: Gene 22 (1983) 229-235; Ulmanen et al.: J. Bacteriol 162 (1985) 176-182; and Meyer and Fiechter: Appl. Environ. Microbiol. 50 (1985) 503-507).
One general problem with organisms that have cloned genes is the low expression of foreign or abnormal proteins. Gene expression depends on environmental conditions which determine cellular physiology. In many cases, optimal culture conditions for product formation are not identical with optimal growth conditions, and in some cases the foreign gene products may even be toxic to the host organism.
The results from the attempts to express foreign genes in bacilli mentioned above have indicated that either the regulation is performed on the translational level, or the foreign gene product is decomposed through the proteolytic action from exoenzymes native to B. subtilis. The yields of foreign proteins have therefore in these attempts been very variable.
In E. coli this problem has been alleviated by the use of inducible operator-repressor expression systems as those mentioned above.
Use of the lac repressor-operator from E. coli in B. subtilis has been reported by Yansura and Henner (Proc. Natl. Acad. Sci. U.S.A. 81 (1984) 439-443). This solution, however, suffers from the disadvantage that the lac operator-repressor system is not native to bacilli.
For a number of years it has been known that the production/occurrence of a number of enzymes in bacilli could be induced by xylose and other sugars in the growth medium, but the mechanism behind this phenomenon has remained unknown. The enzymes induced by xylose are those necessary for xylan utilization in bacilli such as described by I. G. Roncero, J. Bacteriol. 156 (1983) 257-263.
For a number of years it has also been known that this xylose induction only occurs when a suitable growth phase has been obtained by the culture, i.e., when the logarithmic growth phase has ceased and the culture has reached a certain maturity.
The structural gene for the production of xylanase has been cloned and used for the expression of xylanase in E. coli and B. subtilis (Japanese published patent No. 75286-A and 9198978-A).